弗氏柠檬酸杆菌感染诱导斑马鱼皮肤免疫相关基因的差异表达

从草鱼肠道中分离到1株细菌,通过形态学观察、生理生化特征和16S rDNA序列分析等鉴定为弗氏柠檬酸杆菌,动物试验结果表明,该菌对斑马鱼有致病性;从感染诱导的斑马鱼皮肤组织中提取总RNA,经Biotin荧光标记与拥有15 617个cDNA片段的斑马鱼基因芯片(affymetrix)杂交筛选分析,获得斑马鱼皮肤免疫相关差异表达基因88个,其中有74个上调表达基因和14个下调表达基因;进一步根据基因功能聚类(GO)分析,初步将88个差异表达基因分为8个生物学功能,其中主要参与补体激活、急性期反应、应激防御反应、细胞凋亡、抗原加工提呈、细胞迁移粘附、凝血因子和血小板激活等免疫应答过程;同时进行信号通路(pathway)分析,结果表明MAPK(hsp70、daxx、nfkbiab)、JAK/STAT(ifn1)和TGF-β(thbs1)等信号通路参与斑马鱼皮肤抗弗氏柠檬酸杆菌感染免疫应答。采用基因芯片方法筛选与鱼类皮肤免疫相关的功能基因,为将来以斑马鱼为模型研究鱼类皮肤局部免疫应答的分子机制提供科学依据。

Differential expression of immune-related genes from skin of zebrafish induced by the infection of Citrobacter freundii

With water pollution aggravated and intensive aquaculture spread,the occurrence of opportunistic infections has become a significant health problem in farmed fish.Citrobacter freundii is an enterobacterium commonly isolated from diseased and healthy animals,including human,mammals and fish,which is considered to be an opportunistic pathogen.Skin is an essential protective barrier for fish and functions as a first line of defense against invading pathogens,but its molecular mechanism remains unclear.The objective of this study is to screen the differential gene expression in response to pathogen infection in skin of fish,and provide a basis for understanding the fish skin immune mechanism to the pathogen at the molecular level.Here,a strain of bacteria was isolated from intestine in Ctenopharyngodon idellus,which was identified by using the morphological observation,physiological and biochemical characteristics,and 16S rDNA sequence analysis.The results showed that the isolated strain was identified species levels as Citrobacter freundii,and animal tests showed that the strain was lethal to zebrafish.Immune response in skin of fish analyzed by using Citrobacter freundii induced zebrafish as a model,which applied the Affymetrix zebrafish cDNA microarray hybridized to the skin tissues.Total RNAs were isolated from the skin tissues of the zebrafish and labeled with biotin,and hybridized to zebrafish cDNA gene chips.The expression profiles from the hybridization to 15617 genes in zebrafish cDNA array were analyzed by the GeneChip Operating Software(GCOS 1.4).Out of 15617 genes in zebrafish cDNA chips,total of 88 immune-related genes were identified to be significantly expressed in the skin tissues,of which 74 genes are up-regulated and 14 are down-regulated in the zebrafish skin tissues.Furthermore,the differential expression genes could be categorized into eight functional groups by using the Gene Ontology(GO)method,including complement activation,acute phase response,defense immune response,platelet activation,coagulation factors,response to stress and stimulus,apoptosis,antigen processing and presentation,cell adhesion and migration,etc.In addition,the analysis of the pathways showed that the MAPK(hsp70,daxx,nfkbiab),JAK/STAT(ifn1),and TGF-β(thbs1) signaling pathways may be involved in the immune responses of skin in fish.The Affymetrix zebrafish cDNA microarray is useful for identifying the functional genes involved in the skin immune defense of fish,and future functional analysis of these genes may contribute to understanding the mechanisms of skin immune responses and offer a scientific reference for the Citrobacter-zebrafish model of bacterial pathogenesis.